Methods to preserve biologic materials in storage have a long history, from the preservation of food to the preservation of modern pharmaceutical compositions. Biological materials have been dried, salted, frozen, cryoprotected, spray dried, and freeze-dried. Optimal methods of preservation can depend on the acceptable degree of degradation, the desired storage time, and the nature of the biological material.
For centuries, food has been preserved for later consumption by drying. Food harvested in times of plenty was laid out in the sun to remove excess water. Drying can make the food unsuitable for growth of spoilage bacteria and fungi. Autolytic processes, in which plant and animal tissues self destruct, can also be prevented by drying. Salting food can provide a similar preservative effect. Dried and salted food usually experiences a loss of fresh appearance and nutritional value. Drying and salting bioactive materials, such as enzymes and pharmaceuticals, destroy activity by heat, oxidation, water removal, production of radicals and peroxides, photobleaching, and the like, that denature the material.
Spray drying has been used in food processing and pharmaceutical production with some advantages over salting or slow drying. Water can be quickly removed by spraying a fine mist of the dissolved biological molecules into a stream of hot gasses. The dried particles can have a large surface to volume ratio for speedy reconstitution with aqueous buffers. In Platz et al., U.S. Pat. No. 6,165,463, “Dispersible Antibody Compositions and Methods for Their Preparation and Use”, for example, dry powder particles are prepared by spray drying for inhaled administration of pharmaceuticals to patients. The biological molecules, in a dilute solution, are sprayed at moderate pressures (e.g., 80 psi) into a stream of hot gasses (e.g., 98-105° C.) for primary drying, then the particles are further dehydrated by prolonged exposure to high temperatures (e.g., 67° C.). Although such processes are suitable for food and rugged biomolecules, sensitive molecules can be denatured, or otherwise inactivated, by the stress, long drying periods, and high temperatures of these methods.
Freezing can be an effective way to preserve biological molecules. Cold temperatures can slow degradation reaction kinetics. Freezing can reduce the availability of water to degradation reactions and contaminant microbes. Ice can reduce oxidation of the molecules by blocking contact with air. However, freezing can have negative effects such as concentration of salts that can denature proteins in the unfrozen solution, or the formation of sharp ice crystals that can pierce cell structures. Some of the damage caused by freezing can be mitigated by the addition of cryoprotectants which prevent denaturation by lowering the freezing temperature and inhibiting formation of ice crystals. Even in cases where freezing and thawing degradation can be avoided, continuous operation of refrigeration equipment can make preservation by storage in a freezer inconvenient and expensive.
Freeze-drying processes have many of the benefits of freezing and drying. Degradation is suspended by freezing then water removal makes the product more stable for storage. Drying by sublimation of the frozen water into a vacuum can avoid the high heat of some spray drying processes. The lyophilized product can be quite stable in storage even at room temperatures. However, the molecules can still experience denaturing salt concentrations during the freezing step. In addition, many freeze-drying protocols call for prolonged secondary drying steps at high temperatures to reduce moisture content. Bulky cakes of lyophilized material can be slow to reconstitute and must be finely ground for delivery by inhalation.
A need remains for compositions and methods to prepare stable particles containing bioactive materials without loss of purity due to excessive heat, chemical, or shear stress. The present invention provides these and other features that will become apparent upon review of the following.